A. Field Of The Invention
This invention relates to an improved method for synthesizing sulfur and selenium containing phosphoanhydrides. It also relates to a unique family of cyclo-intermediates created during this synthesis. The invention is especially well suited for synthesizing nucleoside thiotriphosphates such as adenosine-5'-[2-thiotriphosphate].
B. Description Of The Art
Because of the important function that nucleotides and nucleosides have in the genetic make up of the body, they have been the subject of much study. Nucleotides can be defined as an organic compound having a nitrogenous base, a five carbon backbone (usually a sugar), and a phosphoric acid. Nucleosides are similar in structure, except that the phosphate group is not present.
Many nitrogenous bases are derived from purine and pyrimidine such as uracil, thymine, cytosine, 5-methyl cytosine, 5-hydroxy-methyl cytosine, adenine, guanine, 2-methyladenine, 1-methylguanine. Nucleosides are usually N-glycosides of these pyrimidine or purine bases. Among these are the ribonucleosides which contain D-ribose as the sugar component, and the 2' deoxyribonucleosides which contain 2'-deoxy-D-ribose as the sugar component. The most prevalent nucleosides are adenosine, guanosine, cytidine, uridine, 2' deoxyadenosine, 2' deoxyguanosine, 2' deoxycytidine, and 2' deoxythymidine.
The names for the corresponding nucleotides are the same except that "5'-phosphoric acid" is added. The nucleotides are also known by their abbreviations AMP, GMP, CMP, UMP, dAMP, dGMP, dCMP, and dTMP. These nucleotides also occur as the 5' di-phosphates and the 5' triphosphates (e.g. ADP, ATP).
A very important 5' tri-phosphate is adenosine 5' triphosphate ("ATP"). Its sulfur variant, adenosine-5' [2-thiotriphosphate] ("ATPBS") has been widely used in stereochemical studies of phosphotransferases and ATP-dependent synthetases. F. Eckstein and R. S. Goody, 15 Biochemistry 1685-1691 (1976); E. K. Jaffe and M. Cohn, 17 Biochemistry 652-657 (1978), 253 J. Biol. Chem. 4823-4825 (1978) and 254 J. Biol. Chem. 10839-10845 (1979). (The disclosures of these articles and of all other articles cited herein are incorporated by reference as if fully set forth herein.) Other variants of ATP (or other nucleoside tri-phosphates) where there has been substitution of one, two, three, or four sulfur or selenium for the phosphate-linked oxygen are also of interest. Also, it is of interest to create other types of triphosphate compounds that have selenic phosphate and thiophosphate groups, e.g. oligonucleotide thio-triphosphates.
A prior art synthesis of ATPBS was reported by R. S. Goody and F. Eckstein, 93 J. Am. Chem. Soc. 6252-6257 (1971), and Eckstein & Goody (1976) supra. They adapted a general procedure introduced by Michelson for synthesizing phosphoanhydrides, which is reported in A. M. Michelson, 91 Biochem. Biophys. Acta 1-13 (1964), to the preparation of sulfurcontaining nucleoside phosphoanhydrides. However, the overall yield of this process was low (12% for ATPBS). It can therefore be seen that it would be desirable to have a low cost procedure for making ATPBS and related sulfur and/or selenium containing triphosphate compounds in high yields.